Anna M. GOMEZ-FOIX,* Joan E. RODRIGUEZ-GIL,t Joan J. GUINOVART* and Fatima BOSCHt. * Department of Biochemistry and Physiology, University of ...
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Biochem. J. (1991) 274, 309-312 (Printed in Great Britain)
Prostaglandins E2 and F2X increase fructose 2,6-bisphosphate levels in isolated hepatocytes Anna M.
GOMEZ-FOIX,* Joan E. RODRIGUEZ-GIL,t Joan J. GUINOVART* and Fatima BOSCHt
Department of Biochemistry and Physiology, University of Barcelona, Marti i Franques 1, 08028-Barcelona, and t Department of Biochemistry, Autonomous University of Barcelona, School of Veterinary Medicine, 08193-Bellaterra (Barcelona), Spain *
In hepatocytes isolated from fed rats, prostaglandin E2 (PGE2) and prostaglandin F2. (PGF2,) increased, in a time- and dose-dependent manner, fructose 2,6-bisphosphate [Fru(2,6)P2] levels and stimulated the glycolytic flux. The rise in Fru(2,6)P2 was related to an increase in glucose 6-phosphate levels which resulted from the stimulation of glycogenolysis. In cells obtained from 24 h-starved rats, no effects of either PGE2 or PGF2 could be observed. In addition, when the stimulation of glycogenolysis was abolished by incubation of fed-rat hepatocytes in a Ca2+-depleted medium, Fru(2,6)P2 levels did not increase. Furthermore, no effects of PGs on 6-phosphofructo-2-kinase activity could be observed. These results indicate that PGE2 and PGF2a show similar actions to Ca2+-dependent hormones on hepatic glucose metabolism. 2a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2
INTRODUCTION The metabolism of glucose and glycogen in the liver is under a complex hormonal control. Apart from the well-known glycogenolytic effectors [glucagon, adrenaline (epinephrine), angiotensin, vasopressin and oxytocin] (see [1] for a review), an additional group of glycogenolytic agents, whose effects appear in perfused liver but fail to appear in isolated hepatocytes, has been described. This group includes the phorbol ester phorbol 12-myristate 13-acetate (PMA; 'TPA') and the platelet activating factor (PAF) [2-4]. Their action on glycogen metabolism involves non-parenchymal liver cells secreting mediators modifying the metabolism of hepatocytes. Several studies have identified prostaglandins (PGs) as the intercellular mediators, inside the liver, of the glycogenolytic action of these effectors [3,5-8]. PGs have been shown to be able to regulate hepatic glycogen metabolism. Administration of PGE1 induced an inactivation of glycogen synthase [9]. In isolated hepatocytes, direct effects of PGs on glycogen metabolism have also been observed. PGD21 PGE2 and PGF2. stimulated glucose production [10]. PGE2 and PGF2. inactivated and phosphorylated glycogen synthase and activated glycogen phosphorylase [5,8,11,12]. These effects of PGs were dependent on the presence of Ca21 in the incubation media [11]. In this regard it has been observed that PGE2 and PGF2X increase intracellular Ca2+ levels [10] by stimulating the production of Ins(1,4,5)P3 [13]. Previous studies showed that PGs acted on liver glycogen metabolism in a similar manner to Ca2+-dependent hormones. The present paper examines the effects of PGE2 and PGF2. on the glycolytic pathway in isolated hepatocytes. We report that these PGs are able to stimulate the glycolytic flux effect that is probably the result of an increase in Fru(2,6)P2 levels. The increase in Fru(2,6)P2 can be related to the stimulation of glycogenolysis.
MATERIALS AND METHODS Materials PGE2 and PGF2a, glucagon, [Arg8]vasopressin and oxytocin were from Sigma. All other chemicals were of analytical grade. Stock solutions of PGs were made in dimethyl sulphoxide (DMSO).
Preparation and incubation of hepatocytes Parenchymal liver cells were isolated from 24 h-starved or fed male Wistar rats, essentially by the method of Berry & Friend, modified as previously described [14]. Hepatocytes were resuspended in a Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 02/CO2 (19: 1). When stated, Ca2+ was omitted from this medium and 1 mM-EGTA was added. Enzyme and metabolite assays For the measurement of Fru(2,6)P2 concentrations and 6phosphofructo-2-kinase activity, 0.1 ml samples of cell suspension were frozen in liquid N2. To determine Fru(2,6)P2, frozen samples were thawed in 0.6 ml of 54 mM-NaOH and, after heating at 80 °C for 5 min, Fru(2,6)P2 was measured by its ability to activate PP1: fructose 6-phosphate 1-phosphotransferase as described in [15]. Activity of 6-phosphofructo-2-kinase was measured as described in [16]. To determine glucose 6-phosphate, 2 ml samples of hepatocyte suspension were centrifuged (3000 g, 20s), and cell pellets were immediately homogenized with 0.5 ml of ice-cold 10% (w/v) HC104. Glucose 6-phosphate concentration was measured enzymically in neutralized HC104 extracts [17]. Glucose and L-lactate concentrations were measured in the 12000 g cell supernatants. Glucose was quantified using the hexokinase method (Gluco-Quant System; Boehringer Mannheim). Lactate was measured enzymically [18]. Glycogen phosphorylase activity was determined as previously described [11]. RESULTS Effects of PGE2 and PGF2,on Fru(2,6)P2 concentration When hepatocytes, isolated from fed rats and incubated with 8 mM-glucose, were treated with 1 ,#M-PGE2 or -PGF2., a marked increase in Fru(2,6)P2 levels was observed. The increase caused by both effectors was similar and reached a maximum after 5 min of incubation (Fig. la). Values then decreased and returned to basal levels after 75-90 min. This behaviour was similar to that of maximal concentrations of vasopressin (0.1 M) and oxytocin (1 /M), which also provoked a transient increase in Fru(2,6)P2 levels (Fig. lb), although vasopressin was more potent than oxytocin. However, when hepatocytes were maintained in a Ca2+-depleted medium containing 1 mM-EGTA, neither PGE2
Abbreviations used: PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate ('TPA'); PAF, platelet-activating factor; DMSO, dimethyl sulphoxide; Fru(2,6)P2, fructose 2,6-bisphosphate.
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Fig. 1. Effect of PGE2. PGF2,. vasopressin and oxytocin on Fru(2,6)P2 levels in bepatocytes isolated from fed rats (a) Hepatocytes prepared from fed rats were incubated with KrebsRinger bicarbonate buffer containing 8 mM-glucose, in the presence of 2.5 mM-Ca2+ (A, *, 0) or in the absence of CaCl2 plus 1 mMEGTA (A, O, 0). After 30 min of preincubation, cells were treated with 1 1sM-PGE2 (, O), 1 uM-PGF2. (A, A) or their solvent (0, 0), for different times. Values are means +S.E.M. for nine experiments performed in different cell preparations and are expressed per g wet wt. of cells. (b) Hepatic cells from fed rats were incubated as indicated in (a), in the presence of 2.5 mM-Ca2+. After 30 min of preincubation, hepatocytes were incubated with 0.1 #lM-vasopressin (*), 1 /SM-oxytocin (V) or their solvent (0). Results are means + S.E.M. for five separate experiments.
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Fig. 3. Effects of PGE2, PGF2g,, Vasopressin and glucagon on Fru(2,6)P2 levels in hepatocytes from starved rats Parenchymal hepatic cells from 24 h-starved rats were resuspended in a Krebs-Ringer medium with 2.5 mM-Ca2' and 8 mM-glucose. After 30 min of preincubation, hepatocytes were treated for different periods of time with 1 /zM-PGE2 (U), 1 /SM-PGF2. (A)' 0.1 #Mvasopressin (*), 10 nM-glucagon (*) or 1 % DMSO (0). Results are means + S.E.M. for five separate experiments.
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log{[PGJ (M)I Fig. 2. Dose-response of PGE2 and PGF2, to increase Fru(2,6)P2 levels in bepatocytes isolated from fed rats After 30 min preincubation with 8 mM-glucose, parenchymal liver cells isolated from fed rats were treated for 10 min with different concentrations of PGE2 (-) or PGF2a (A). Results shown are means+S.E.M. for five different experiments and are expressed per g wet wt. of cells.
nor PGF2 induced a change in the Fru(2,6)P2 concentration (Fig. la), suggesting that their action is mediated by Ca2+. The effect on Fru(2,6)P2 was also dependent on the concentration of PG used. The half-maximal dose was approx. 0.1 /SM for both PGE2 and PGF2. (Fig. 2). This effect on Fru(2,6)P2 levels appeared to be related to the availability of glycogen in the cells since, in hepatocytes obtained from 24 h-starved rats, no effects of PGE2 or PGF2a on Fru(2,6)P2 levels could be observed. We tested the effects of both PGs in hepatocytes from starved rats incubated under three different conditions: (a) with 8 mm-glucose [Fru(2,6)P2 basal level 6.9 nmol/g] (Fig. 3); (b) with 16 mM-lactate/4 mM-pyruvate (basal level 4.6 nmol/g); and (c) in the absence of any exogenous substrate (basal level 0.33 nmol/g). Under these three conditions neither 1 /tM-PGE2 nor 1 ,#M-PGF2. increased significantly the hepatocyte content of Fru(2,6)P2 (Fig. 3 and results not shown).
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Fig. 4. Effects of PGE2,PGF22 , vasopressin and glucagon on 6phosphofructo24kinase activity in hepatocytes from fed rats Hepatic cells from fed rats were resuspended in a Krebs-Ringer medium containing 2.5 mM-Ca"+ and 8 mM-glucose. After 30 min of preincubation, cells were treated with I 1m-PGE (-), 1 /tM-PGF2. (A), 0.1 M-vasopressin (*), 10 nM-glucagon (*) or 1% DMSO (0) for the indicated times. Values are means+S.E.M. for five independent experiments.
When cells, under the same conditions, were incubated with vasopressin, no effect on Fru(2,6)P2 levels was observed. However, glucagon provoked a profound decrease in Fru(2,6)P2 content (Fig. 3 and results not shown). In order to clarify the mechanism by which PGs increased basal concentrations of Fru(2,6)P2, we next studied whether PGE2 and PGF2, modified 6-phosphofructo-2-kinase activity, the enzyme responsible for Fru(2,6)P2 synthesis. In this regard we 1991
Prostaglandins E2 and
F2. increase fructose 2,6-bisphosphate levels
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of 30 min with 8 mM-glucose, in the were exposed to 1 % 1 DMSO (control), 1 #uM-PGE2 or 1 guM-PGE2 or ,lM-PGF2, for 5 min. Values are means+ S.E.M. for four independent experiments.
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found that treatment of hepatocytes isolated from fed rats with 1 /tM-PGE2 or -PGF2. (from 2 to 60 min) did not affect 6phosphofructo-2-kinase 'a' (active form) (Fig. 4) or total activity (results not shown). Similarly, vasopressin did not modify the activity of this enzyme, whereas incubation with glucagon caused its inactivation (Fig. 4). We next examined whether the accumulation of Fru(2,6)P2 could be related to an increase in the intracellular levels of glucose 6-phosphate. When cells obtained from fed rats were treated with 1 1tM-PGE2 and -PGF2., an increase in the concentration of glucose 6-phosphate was observed. The effect was time-dependent and reached a maximum (up to 50% increase) after 5-10 min of incubation (Fig. Sa). After 30 min of incubation, glucose 6-phosphate values were about 20 % over the control and remained steadily elevated up to 90 min (Fig. Sa). As expected, vasopressin and oxytocin behaved similarly to PGs. Again, vasopressin was more potent than oxytocin (Fig. Sb). In contrast, when cells were incubated in a medium in the absence of Ca2+, glucose 6-phosphate levels did not change in response to PGs (Fig. Sa). Vol. 274
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Fig. 7. Effect of PGE2 and PGF2., on glucose output and lactate production in hepatocytes prepared from fed rats Hepatocytes were preincubated for 30 min and further incubated with 1 % DMSO (@), 1 1sM-PGE2 (M) or 1 /tM-PGF2a (-). Values are means + S.E.M. for three different cell preparations and are expressed per g wet wt. of cells. The statistical significance of the differences in glucose concentration, calculated by the paired t test, was P < 0.05 for both PGs versus control cells.
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Fig. 5. Effect of PGE2, PGF2., vasopressin and oxytocin on glucose 6phosphate concentration in hepatocytes prepared from fed rats (a) Hepatocytes were preincubated for 30 min in a Krebs-Ringer bicarbonate buffer with 8 mM-glucose, in the presence (A, *, 0) or absence of CaCl2 (A, r, 0) and further incubated for different times with 10% DMSO (0, 0), 1 ,zM-PGE2 (M, rO) or 1 #ma-PGF2. (A, A). Results are means+ S.E.M. for nine cell preparations and are expressed per g wet wt. of cells. (b) Hepatocytes resuspended as described in (a) with 2.5 mM-Ca2" were treated with 0.1 LM-vasopressin (*), 10 nM-oxytocin (V) or their solvent (-), after 30 min of preincubation, during the indicated times. Results are the mean + S.E.M. for five separate experiments.
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Effects of PGE2 and PGF2. on glycogenolysis and lactate production Since we had previously shown that PGE2 and PGF2. inactivated glycogen synthase and activated glycogen phosphorylase in hepatocytes from starved rats [11], we investigated whether the glucose 6-phosphate accumulation in hepatocytes from fed rats could be deleted to the activation of glycogen phosphorylase. In hepatocytes from fed rats, both PGs, PGE2 and PGF22, caused a 2-fold increase in glycogen phosphorylase activity in a manner similar to that in hepatocytes from starved rats. However, in cells incubated in a medium without Ca2 , in which lower basal levels were observed, no activation was obtained after treatment with either PGE2 or PGF2. (Fig. 6). In keeping with the activation of glycogen phosphorylase by PGs, we observed that treatment of hepatocytes from fed rats with PGE2 and PGF2,induced an increase in the glucose output (Fig. 7). As can be observed in Fig. 7, concomitantly with the stimulation of glycogenolysis, PGE2 and PGF2a increased the production of lactate in hepatocytes isolated from fed rats. The increase was already significant after 20 min of incubation. This stimulation of glycolytic flux was in agreement with the observed increase in Fru(2,6)P2. DISCUSSION In the present paper we report that PGE2 and PGF2,stimulate hepatocyte glycolytic rate, as indicated by the increase in lactate production. This effect on glycolytic flux correlates with an increase in the intracellular concentration of Fru(2,6)P2. The mechanism responsible for the effect of PGE2 and PGF22 does not appear to involve covalent modification of 6-phosphofructo-2-kinase, since the activity of this enzyme is not modified
A. M. Gomez-Foix and others
312 in a stable manner by PGs. This behaviour is similar to that observed for vasopressin. The action of prostaglandins on Fru(2,6)PJ content of isolated hepatocytes is related to the availability of glycogen. In hepatocytes from starved rats, even in cells incubated in the presence of different exogenous substrates such as glucose or lactate/pyruvate, PGE2 and PGF2,fail to raise the intracellular levels of Fru(2,6)P2. Similarly, Ca2+-dependent agents only accumulate Fru(2,6)P2 in glycogen-rich hepatocytes (the present paper and [19]). Moreover, we observed a relationship between the activation of glycogen phosphorylase and the accumulation of Fru(2,6)P2. We have previously reported that, in hepatocytes from starved rats [11], the effects of PGE2 or PGF2, on glycogen-metabolizing enzymes were mediated by Ca2+. Likewise, we observe here that, in cells obtained from fed rats, the activation of glycogen phosphorylase by PGE2 or PGF2, only occurs when hepatocytes are incubated in the presence of Ca2 . We also show that, in hepatocytes prepared from fed rats and incubated in a Ca2+_ depleted medium, in which glycogen phosphorylase is not activated by PGE2 or PGF2., neither glucose 6-phosphate nor Fru(2,6)P2 was increased as in response to PGs. Again, the effects of these PGs strongly remind one of the action of Ca2+dependent agents such as phenylephrine, vasopressin and also the Ca2+ ionophore A23187 [19], which do not increase the Fru(2,6)P2 content of isolated hepatocytes when glycogen phosphorylase is inactivated. These results therefore suggest that the increase in Fru(2,6)P2 is related to the increase in glucose 6-phosphate levels, which results from the stimulation of glycogenolysis. However, other explanations, such as a non-covalent activation of 6-phosphofructo-2-kinase, cannot be disregarded. In conclusion, our data support the hypothesis that PGE2 and PGF2a show similar effects to Ca2+-dependent hormones in hepatocytes, namely their ability to stimulate glycogenolysis and glycolysis and to oppose the effects of glucagon. We thank Ms. Anna Vilalta and Ms. Catalina Relafio for their valuable technical assistance. This work was supported by a grant from
the Fondo de Investigaciones Sanitarias de la Seguridad Social (n° 90/0301). J.E.R.-G. was the recipient of a fellowship from the Formaci6n de Personal Investigador (Ministry of Education, Spain). Ms. Catalina Relaino was recipient of a fellowship from the Fondo de Investigaciones Sanitarias de la Seguridad Social.
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Received 9 April 1990/22 November 1990; accepted 17 December 1990
1991
